Liver stiffness diminishes with antiviral response in chronic hepatitis C.

Liver stiffness diminishes with antiviral response in chronic hepatitis C. Christophe Hézode, Laurent Castéra, Françoise Roudot-Thoraval, Magali Bouvier-Alias, Isabelle Rosa, Dominique Roulot, Vincent Leroy, Ariane Mallat, Jean-Michel Pawlotsky To cite this version: Christophe Hézode, Laurent Castéra, Françoise Roudot-Thoraval, Magali Bouvier-Alias, Isabelle Rosa, et al.. Liver stiffness diminishes with antiviral response in chronic hepatitis C.: Liver Stiffness Kinetics on HCV Therapy. Alimentary Pharmacology and Therapeutics, Wiley,, (), pp.-. <./j.-..0.x>. <inserm-00> HAL Id: inserm-00 http://www.hal.inserm.fr/inserm-00 Submitted on Sep HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Alimentary Pharmacology & Therapeutic Liver Stiffness Diminishes with Antiviral Response in Chronic Hepatitis C Journal: Alimentary Pharmacology & Therapeutics Manuscript ID: APT-00-.R Wiley - Manuscript type: Original Scientific Paper Date Submitted by the Author: n/a Complete List of Authors: Hezode, Christophe Castéra, Laurent Roudot-Thoraval, Françoise Bouvier-Alias, Magali Rosa, Isabelle ROULOT, Dominique; Hopital jean verdier, Liver unit Leroy, Vincent Mallat, Ariane Pawlotsky, Jean-Michel; Hopital Henri Mondor - Université Paris XII, Virology Keywords: Hepatology, Hepatitis C < Hepatology, Liver fibrosis < Hepatology, Viral hepatitis < Hepatology

Page of Alimentary Pharmacology & Therapeutic 0 0 0 0 Liver Stiffness Diminishes with Antiviral Response in Chronic Hepatitis C Christophe Hézode,, * Laurent Castéra, * Françoise Roudot-Thoraval,, Magali Bouvier- Alias,, Isabelle Rosa, Dominique Roulot, Vincent Leroy, Ariane Mallat,, and Jean-Michel Pawlotsky, Department of Hepatology and Gastroenterology, Hôpital Henri Mondor, Université Paris-Est, Créteil, France; INSERM U, Créteil, France; Department of Hepatology and Gastroenterology; Hôpital Haut- Lévêque et Hôpital Saint-André, Université Victor Segalen Bordeaux II, Bordeaux, France; Department of Public Health, Hôpital Henri Mondor, Université Paris-Est, Créteil; National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Hôpital Henri Mondor, Université Paris-Est, Créteil, France; Department of Hepatology and Gastroenterology, Centre HospitalierIntercommunal, Créteil, France; Department of Hepatology and Gastroenterology, HôpitalAvicenne, Université Paris, Bobigny, France; Department of Hepatology and Gastroenterology, Hôpital de la Tronche, University of Grenoble, Grenoble, France *These authors equally contributed to the work RUNNING HEAD: Liver Stiffness Kinetics on HCV Therapy Corresponding author: Prof. Jean-Michel Pawlotsky, MD, PhD, Department of Virology, Hôpital Henri Mondor, avenue du Maréchal de Lattre de Tassigny, 0 Créteil, France. Tel: +---; Fax: +---. E-mail: jeanmichel.pawlotsky@hmn.aphp.fr

Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 ABSTRACT Background: Transient elastography measures liver stiffness, which correlates with the hepatic fibrosis stage and has excellent accuracy for the diagnosis of cirrhosis in patients with chronic hepatitis C. Aim: To prospectively assess the kinetics of liver stiffness in treated patients with chronic hepatitis C and compare them with the viral kinetics on treatment and with the final outcome of therapy. Methods: patients with chronic hepatitis C with significant fibrosis (>.0 kpa) at baseline were included. They received therapy with pegylated interferon-α and ribavirin. The kinetics of liver stiffness were characterized during therapy and thereafter by means of Fibroscan, and compared with the virological responses at weeks,,, end of treatment and and weeks after. Results: A significant liver stiffness decrease was observed during therapy, which continued after treatment only in patients who achieved a sustained virological response. In this group, the median intra-patient decrease relative to baseline at the end of follow-up was -. kpa, vs -. kpa in the patients who did not achieve an SVR. Similar dynamics were observed in cirrhotic and non-cirrhotic patients. In multivariate analysis, only the SVR was associated with long-term improvement of liver stiffness (odds ratio:.; % confidence interval:.-.0, p=0.0). Conclusions:In patients with advanced fibrosis at the start of therapy, liver stiffness is significantly reduced during treatment, but improvement continues off treatment only in patients who achieve a sustained virological response. Liver stiffness

Page of Alimentary Pharmacology & Therapeutic 0 0 0 0 assessment earlier than months after the end of therapy does not appear to be clinically meaningful.

Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 Chronic infection with hepatitis C virus (HCV) is the leading cause of chronic liver disease in Europe and the United States. Chronic hepatitis C is responsible for substantial morbidity and mortality related to liver cirrhosis and its complications, including hepatocellular carcinoma. Nowadays, HCV is becoming the first cause of primary liver cancer and is the main indication for liver transplantation in industrialized countries. The current standard treatment of chronic hepatitis C is a combination of pegylated interferon (IFN)-α and ribavirin. With this treatment, a sustained virological response, defined as an undetectable HCV RNA weeks after the end of therapy, is achieved in approximately 0%-0% of patients infected with HCV genotype and in 0% of those infected with genotypes and. - The hepatic fibrosis stage is the principal predictor of liver disease progression and drives treatment indications. Liver biopsy examination has traditionally been considered the reference method for staging liver fibrosis. It is recommended in recent Clinical Practice Guidelines for treatment decision in patients with chronic hepatitis C. However, the accuracy of liver biopsy has been questioned, as sampling errors and intra- and inter-observer variability may lead to under- or over-staging. - In addition, liver biopsy is an invasive procedure with rare, but potentially life-threatening, complications. - These limitations have stimulated the search for noninvasive approaches for liver disease severity assessment, including serological markers and methods based on ultrasonography, such as transient elastography. - Transient elastography by means of the Fibroscan (Echosens, Paris, France) measures liver stiffness. It can be performed at the bedside with immediate results and

Page of Alimentary Pharmacology & Therapeutic 0 0 0 0 has been reported to be rapid, user-friendly and reproducible. Liver stiffness has been shown to correlate with the hepatic fibrosis stage and to have excellent accuracy for the diagnosis of cirrhosis in patients with chronic hepatitis C. - Thus far, transient elastography has been used and validated essentially in cross-sectional studies, whereas the kinetics of liver stiffness in patients with chronic hepatitis C receiving antiviral therapy have not been characterized in prospective longitudinal studies. The goal of this study was to prospectively assess the kinetics of liver stiffness in patients with chronic hepatitis C treated with pegylated IFN-α and ribavirin and to compare them with the HCV RNA level kinetics on treatment and with the final outcome of therapy. PATIENTS AND METHODS Patients This multicentre, prospective study was conducted in French hospitals between January 0 and March 0. One hundred and five patients with chronic hepatitis C, i.e. patients with chronically elevated serum alanineaminotransferase (ALT) levels and detectable serum anti-hcv antibodies and HCV RNA, were included. All of them were treatment-naïve and the main inclusion criterion was significant fibrosis, defined as liver stiffness >.0 kilopascals (kpa) with the Fibroscan at baseline. The exclusion criteria were: a coinfection with hepatitis B virus or human immunodeficiency virus, a daily alcohol intake >0 g, decompensated liver disease or hepatocellular carcinoma, liver transplantation, and a failed or unreliable liver stiffness measurement.

Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 The study protocol conformed to the ethical guidelines of the Helsinski declaration and the French regulations on clinical trials, and was approved by the Institutional Review Board (Comité de Protection des Personnesd Ile-de-France IX). The patients were enrolled after giving their written informed consent. Antiviral therapy The patients received standard-of-care therapy with either pegylated IFN-αa (Pegasys, Hoffman-La Roche, Basel, Switzerland), 0 µg once weekly, or pegylated IFN-αb (PegIntron, Schering-Plough, Kenilworth, New Jersey),. µg/kg once weekly, and ribavirin,.0-. g/day according to body weight below or above kg, respectively, in patients infected with genotypes and, or 0. g/day in patients infected with HCV genotypes and. Treatment duration was weeks in patients infected with HCV genotypes and (with a stopping rule at week if the HCV RNA decline was less than Log international units (IU)/mL), and weeks in patients infected with HCV genotypes and. Monitoring schedule and definition of virological responses ALT levels, HCV RNA levels and liver stiffness were evaluated at baseline, at weeks,, and of therapy in all patients, at week of therapy in patients infected with HCV genotypes and, and and weeks after treatment withdrawal in all patients. On treatment, virological responses were defined as follows: rapid virological response (RVR): undetectable HCV RNA at week ; early virological response (EVR): undetectable HCV RNA at week ; end-of-treatment response (EOTR): undetectable

Page of Alimentary Pharmacology & Therapeutic 0 0 0 0 HCV RNA at the end of treatment, i.e. week or in patients infected with HCV genotypes - and -, respectively. The sustained virological response (SVR) was defined as an undetectable HCV RNA weeks after the end of therapy. HCV RNA level measurement HCV RNA levels were measured centrally by means of the m00 SP /m00 RT real-time PCR platform (Abbott Molecular, Des Plaines, Illinois), according to the manufacturer s instructions. The assay has been shown to accurately quantify HCV RNA levels regardless of the HCV genotype. Its lower limit of detection is IU/mL. Undetectable HCV RNA at the different time points was thus defined as an HCV RNA level < IU/mL. Liver stiffness measurement Liver stiffness measurements were performed with the FibroScan device, as previously described. Ten validated measurements were performed for each patient. The success rate was calculated as the number of validated measurements divided by the total number of measurements. The results were expressed in kilopascals. The median value was considered representative of the elastic modulus of the liver. Only procedures with at least ten successful acquisitions, a success rate of at least 0% and an interquartile range (IQR) of less than 0% of the median value were considered reliable. Patients with baseline liver stiffness values above kpa were considered to have cirrhosis. Long-term improvement of liver stiffness was defined as a decrease of at least 0% of median values months after treatment withdrawal relative to baseline, as the manufacturer allows fluctuations of 0% of the IQR relative to the median value.

Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 Statistical analysis Results are expressed as the mean ± standard deviation for normally distributed variables (age, body mass index [BMI]) and as the median with IQR ( st and rd quartiles) for variables with asymmetric distribution (ALT, HCV RNA level and liver stiffness). Intra-group comparisons were made using Wilcoxon s test for paired data. Categorical data were expressed as numbers and percentages, and compared by means of the Chi-square test or Fisher s exact test where appropriate. The relationship between quantitative data was tested by linear regression analysis. The factors associated with long-term improvement of liver stiffness were tested by univariate analysis (chi-square test, Mann and Whitney test). Variables with a p value 0. were then tested in a logistic regression model by a forward step-by-step procedure. Odds ratios (OR) and their % confidence intervals (CI) were inferred from the model. A p value 0.0 was considered significant. RESULTS Patient characteristics and disposition Among the patients prospectively included in the study, were lost to followup and an additional patients did not complete follow-up (more than missing time points) and were excluded from the analysis. These patients did not differ from the other treated patients for age, gender, BMI, frequency of cirrhosis, HCV genotype, and baseline HCV RNA level, ALT level, and liver stiffness.

Page of Alimentary Pharmacology & Therapeutic 0 0 0 0 Overall, treated patients were analyzed. There were men and women, and their mean age was.±. years. Their characteristics are shown in Table. Among the patients, were treated for weeks only (one patient who did not achieve a -Log HCV RNA drop at week and one who achieved an RVR and stopped therapy at week ); patients infected with genotype or were treated for weeks; and patients infected with genotype, or were treated for weeks. An RVR and an EVR were observed in patients (.%) and patients (.%), respectively. An SVR was observed in patients (.%). Median liver stiffness changes according to the virological response Table shows the median intra-patient changes in liver stiffness relative to baseline in the patients who achieved or did not achieve an SVR. No significant relationship was found between liver stiffness changes and the RVR, EVR or EOTR (data not shown). A significant liver stiffness decrease was observed during pegylated IFN-α and ribavirin administration in the patients who achieved an SVR, as well as in non-sustained virological responders (median changes at the end of treatment relative to baseline: -.0 kpa, p<0.00, and -. kpa, p=0.0, respectively) (Figures A and B, Table ). After treatment, liver stiffness continued to significantly decrease relative to end-of-treatment in the patients who achieved an SVR (median change: -0. kpa, p=0.00), resulting in a median intra-patient decrease relative to baseline at the end of follow-up of -. kpa (p<0.00) (Figure B and Table ). In contrast, in patients who did not achieve an SVR, liver stiffness increased after the end of treatment (median change: +0. kpa, p=0.), resulting in a median intra-patient decrease of liver stiffness relative

Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 to baseline of -. kpa (p=0.0) (Figure A and Table ). However, as shown in Figure, in spite of the global trends, there were important differences in liver stiffness kinetics between individual patients. Liver stiffness changes according to the virological response in cirrhotic patients When cirrhotic and non-cirrhotic patients were considered separately, liver stiffness values were always significantly higher in the former than in the latter, but the same dynamics as in the global analysis were seen. They are presented in Table. Liver stiffness changes according to ALT changes No relationship was observed between liver stiffness and ALT changes during and after therapy, regardless of the virological outcome of treatment (data not shown). Variables associated with long-term improvement of liver stiffness Long-term improvement of liver stiffness was defined as a decrease of median liver stiffness between baseline and end of follow-up of 0% or more. Long-term liver stiffness improvement was observed in patients. Table shows the factors that predicted liver stiffness improvement in univariate analysis. In multivariate analysis, only the SVR was associated with long-term improvement of liver stiffness, with an odds ratio of. (% confidence interval:.-.0, p=0.0). DISCUSSION

Page of Alimentary Pharmacology & Therapeutic 0 0 0 0 Successful therapy with IFN-α or pegylated IFN-α with or without ribavirinhas been shown to be associated with significant histological improvements in studies in which patients underwent paired liver biopsies. - However, follow-up liver biopsy is not part of the routine management of patients with chronic HCV infection receiving antiviral therapy. Therefore, non-invasive methods appear to be better suited to the monitoring of liver disease outcomes during and after therapy. Given its simplicity, high acceptability by patients and intrinsic performance. -, 0, transient elastography is an appropriate tool for the longitudinal follow-up of fibrosis changes in treated HCV-infected patients, as shown in the present study. In this multicentre study conducted in expert centres for transient elastography and HCV virology, we characterized the dynamics of liver stiffness in patients with significant fibrosis at baseline (defined as liver stiffness >.0 kpa with the Fibroscan ) treated with pegylated IFN-α and ribavirin. Liver stiffness and HCV RNA kinetics were assessed at multiple time points during and after therapy and compared. We observed that liver stiffness significantly decreased on treatment both in patients who did and did not achieve an SVR. However, liver stiffness continued to significantly decrease after the end of treatment only in patients who achieved an SVR. On treatment, no relationship was found with RVR, EVR or EOTR. Overall, both patients who achieved and those who did not achieve an SVR experienced a significant decrease of liver stiffness at the end of follow-up relative to baseline, but this decrease was greater in the sustained virological responders (p=0.). These results are in keeping with a previous observation in patients who were assessed at the end of treatment and thereafter. The use of mean values for a non-normally distributed variable such as liver stiffness was however

Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 questionable in this study, as median values better reflect the distribution and extreme values. In another study, a significant liver stiffness decrease was reported at the end of follow-up in patients treated with pegylated IFN-α and ribavirin, regardless of the final virological outcome of therapy (SVR or non-svr). No on-treatment monitoring was available, and liver stiffness at baseline significantly differed among the study groups. However, the latter was also the case in our study, with median values of kpa (interquartile range, IQR:.-.) in non-svr vs kpa (.-.) in SVR patients. Overall, our prospectively generated data suggest that the cure of HCV infection is associated with a significant reduction of liver stiffness, as measured by transient elastography. This result may appear surprising as transient elastography is claimed to assess essentially the fibrotic component of liver lesions, while treatment has been mostly associated with reduction of the inflammatory reaction in the liver. Two nonmutually exclusive hypotheses can be raised to explain this result: (i) liver stiffness is also influenced by the local inflammatory reaction; (ii) fibrosis significantly regresses on treatment and thereafter in patients who achieve an SVR. The first hypothesis may appear unlikely as no relationship was observed between liver stiffness changes and ALT kinetics; however, ALT elevations do not always accurately reflect liver inflammation. On the other hand, SVR was the sole predictor of long-term liver stiffness improvement; nevertheless, SVR is also associated with an improvement of liver inflammation. Therefore, this hypothesis cannot be definitively ruled out. Indeed, others have reported an overestimation of liver stiffness in patients with liver inflammation reflected by high ALT levels., - In addition, a significant decrease of liver stiffness was observed during therapy, followed by an increase after treatment withdrawal, in the

Page of Alimentary Pharmacology & Therapeutic 0 0 0 0 patients from this study who did not clear HCV infection. Histological data might have helped resolve this question. However, liver biopsy can no longer be performed during or after antiviral treatment for ethical reasons. Therefore, this question will remain unanswered. Our hypothesis concerning liver stiffness changes in patients who achieved an SVR witnessing fibrosis regression is in keeping with the reported improvement of histological fibrosis in a similar population who underwent paired liver biopsies. - It is also in keeping with the lower incidence of clinical outcomes, including liver failure, variceal bleeding or hepatocellular carcinoma, in patients with cirrhosis who achieved an SVR. - However, although patients with cirrhosis who achieved an SVR experienced a significant improvement of their liver stiffness months after the end of treatment in our study, their median liver stiffness values remained significantly higher than in patients without cirrhosis who achieved an SVR. In this context, the fact that the risk of developing HCC remains substantial in cirrhotic patients who achieved an SVR.,, 0 is not surprising. In conclusion, the results of this prospective, multicentre study show that transient elastography can be used to monitor liver stiffness changes during and after antiviral therapy in patients with chronic hepatitis C. On average, in patients with advanced fibrosis at the start of therapy (>.0 kpa), liver stiffness is significantly reduced during pegylated IFN-α and ribavirin administration. The significant reduction of liver stiffness continues off treatment only in patients who achieve an SVR. This probably denotes fibrosis regression, although it cannot be ruled out that improvements in the inflammatory reaction in these patients may also influence liver stiffness changes. These

Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 findings suggest that assessing liver stiffness by means of transient elastography at baseline and months after treatment in patients who achieve an SVR is useful to assess the global response to antiviral therapy, establish a prognosis and serve as a basis for subsequent follow-up in patients with advanced fibrosis, especially those with cirrhosis. In the latter, post-treatment liver stiffness assessments should not be substituted to the recommended periodic surveillance of hepatocellular carcinoma occurrence based on ultrasound examination and α-fetoprotein level measurement. Our data also suggest that liver stiffness assessment earlier than months after the end of therapy is not clinically meaningful.

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Alimentary Pharmacology & Therapeutic Page of 0 0 0 0 FIGURE LEGENDS Figure. Median (IQR) intra-patient liver stiffness changes relative to baseline at different time points on treatment and after the end of therapy. (A) Patients who did not achieve an SVR. (B) Patients who achieved an SVR. Figure. Individual liver stiffness changes during and after treatment in patients who did not achieve an SVR and in patients who achieved an SVR. EOT: end of treatment; EOF: end of follow-up.

Page of Alimentary Pharmacology & Therapeutic 0 0 Table.Baseline characteristics of the patients. Treated (N = ) Male gender [n (%)] (.) Age (yrs) [mean±sd].±. BMI (kg/m ) [mean±sd].±. ALT (IU/mL) [median (IQR)] (-) HCV genotype [n (%)] -- () - () HCV RNA level (Log IU/mL) [median (IQR)]. (.-.) Liver stiffness (kpa) [median (IQR)]. (.-.) Cirrhosis, i.e. liver stiffness. kpa [n (%)] (.)

Alimentary Pharmacology & Therapeutic Page of 0 0 Table. Median (IQR) intra-patient liver stiffness changes relative to baseline on treatment and thereafter in patients who did and did not achieve an SVR. Treatment duration was weeks in patients infected with HCV genotype or, weeks in patients infected with HCV genotype, or. P values are for intra-patient liver stiffness changes relative to baseline. On treatment Post-treatment Time point SVR (N = ) No SVR (N = ) Liver stiffness change p Liver stiffness change Week -.0 (-. - +0.) 0.00 -. (-. - +0.) 0.0 Week -. (-.0 - +0.) <0.00 -. (-. - +0.) 0.0 End of treatment -.0 (-. - +0.) <0.00 -. (-.0 - -0.) 0.0 Follow-up week -. (-. - +.0) <0.00 -. (-. - +.) 0.0 Follow-up week -. (-. - -.) <0.00 -. (-. - +0.) 0.0 p

Page of Alimentary Pharmacology & Therapeutic 0 0 Table. Median (IQR) intra-patient liver stiffness changes relative to baseline in patients with or without cirrhosis who did or did not achieve an SVR. Treatment duration was weeks in patients infected with HCV genotype or, weeks in patients infected with HCV genotype, or. P values are for intra-patient liver stiffness changes relative to baseline. On treatment Post-treatment Time point SVR (n=) Cirrhosis p No SVR (n=) p SVR (n=0) p No cirrhosis No SVR (n=) Week -. (-. - 0) 0.0 -. (-. - -0.) 0.0-0. (-. - +0.) 0.0 -.0 (-.- +.) 0. Week -.0 (-. - +0.) 0.0 -. (-.0 - +0.) 0.0 -. (-.0 - -0.) 0.00-0. (-.- +.) 0. End of a -.0 (-. - +.) 0.0 -. (-.0 - -0.) 0.0 -. (-.0 - +0.) 0.00 -. (-.- -0.) 0. treatment Follow-up week Follow-up week -. (-. - +.) 0. -. (-. - +.) 0. -. (-. - -0.) <0.00-0. (-.- +.) 0. -. (-. - -.) 0.00 -. (-. - +.) 0. -. (-. - -0.) <0.00 -. (-.- +0.) 0. p

Alimentary Pharmacology & Therapeutic Page of 0 0 Table.Univariate analysis of variables associated with long-term liver stiffness improvement, defined as a liver stiffness decrease 0% relative to baseline weeks after the end of therapy. Liver stiffness change <0% (N = ) Liver stiffness change 0% (N = ) Age (years).±..±. 0. Male gender [n(%)] (.) (.) 0. Baseline BMI (kg/m ).±..±. 0. HCV genotype [n (%)] Genotype -- (.) (.) 0. Genotype - (0.0) (0.0) Baseline ALT level (IU/L) [median (IQR)] (0-) (0-) 0. Baseline liver stiffness (kpa) [median (IQR)]. (.-.). (.-.) 0. Baseline HCV RNA level (Log IU/mL) [median (IQR)]. (.-.). (.-.) 0. Cirrhosis, i.e. liver stiffness > kpa [n (%)] (.) (.) 0. Treatment duration weeks (.) (.) 0. > weeks (.) (.) HCV RNA level weeks after treatment (Log IU/mL) [median (IQR)] 0. (0.-.) 0. (0.-0.) 0.0 Virological response [n (%)] No SVR (.0) (.0) 0.0 SVR (.) 0 (0.) p

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